Eukaryotic gene expression must be regulated such that cells can rapidly respond to a wide range of different conditions. The process of mRNA translation is one step at which gene expression is highly regulated. In response to hormones, growth factors, cytokines and nutrients, animal cells generally activate translation in preparation for the proliferative response. The rate of protein synthesis typically decreases under stressful conditions, such as oxidative or osmotic stress, DNA damage or nutrient withdrawal. Activation or suppression of mRNA translation occurs within minutes and control over this process is thought to be exerted at the initiation phase of protein synthesis (Rosenwald et al., Oncogene, 1999, 18, 2507-2517; Strudwick and Borden, Differentiation, 2002, 70, 10-22).
Translation initiation necessitates the coordinated activities of several eukaryotic initiation factors (eIFs), proteins which are classically defined by their cytoplasmic location and ability to regulate the initiation phase of protein synthesis. One of these factors, eukaryotic initiation factor 4E (eIF4E), is present in limiting amounts relative to other initiation factors and is one component of the eIF4F initiation complex, which is also comprised of the scaffold protein eIF4G and the RNA helicase eIF4A. In the cytoplasm, eIF4E catalyzes the rate-limiting step of cap-dependent protein synthesis by specifically binding to the 5′ terminal 7-methyl GpppX cap structure present on nearly all mature cellular mRNAs, which serves to deliver the mRNAs to the eIF4F complex. Once bound, the eIF4F complex scans from the 5′ to the 3′ end of the cap, permitting the RNA helicase activity of eIF4A to resolve any secondary structure present in the 5′ untranslated region (UTR), thus revealing the translation initiation codon and facilitating ribosome loading onto the mRNA (Graff and Zimmer, Clin. Exp. Metastasis, 2003, 20, 265-273; Strudwick and Borden, Differentiation, 2002, 70, 10-22).
eIF4E availability for incorporation into the eIF4E complex is regulated through phosphorylation as well as through the binding of inhibitory proteins. eIF4E is a phosphoprotein that is phosphorylated on serine 209 by the mitogen-activated protein kinase-interacting kinase Mnk1, as well as by protein kinase C (Flynn and Proud, J. Biol. Chem., 1995, 270, 21684-21688; Wang et al., J. Biol. Chem., 1998, 273, 9373-9377; Waskiewicz et al., Embo J., 1997, 16, 1909-1920). The inhibitory eIF4E-binding proteins 1 and 2 (eIF4E-BP1 and eIF4E-BP2) act as effective inhibitors of translation by competing with eIF4G for binding to the dorsal surface of eIF4E (Ptushkina et al., Embo J., 1999, 18, 4068-4075). When complexed with eIF4E-BP1, eIF4E is not a substrate for phosphorylation by protein kinase C or Mnk1, indicating that dissociation of eIF4E-BP1 from eIF4E is a prerequisite for eIF4E phosphorylation (Wang et al., J. Biol. Chem., 1998, 273, 9373-9377; Whalen et al., J Biol Chem, 1996, 271, 11831-11837). Phosphorylation of eIF4E increases its affinity for mRNA caps, thus elevating translation rates (Waskiewicz et al., Mol. Cell Biol., 1999, 19, 1871-1880).
Fifteen years prior to the cloning of its cDNA, the eIF4E-BP1 protein was identified as a protein phosphorylated in response to insulin and was proposed to be important in insulin action. In addition to insulin, insulin-like growth factor, platelet-derived growth factor, interleukin-2 and angiotensin II also promote the dissociation of eIF4E-BP1 from eIF4E (Lawrence and Abraham, Trends Biochem Sci, 1997, 22, 345-349). eIF4E-BP1 was independently cloned by two strategies, one using amino acid sequence information obtained following purification of the protein, and the other using eIF4E protein to probe a cDNA expression library (Hu et al., Proc Natl Acad Sci USA, 1994, 91, 3730-3734; Pause et al., Nature, 1994, 371, 762-767). eIF4E-BP1 is also known as phosphorylated heat- and acid-stable protein regulated by insulin (PHAS-I). eIF4E-BP1 is expressed in most human tissues, including heart, brain, placenta, lung, liver, kidney and spleen, and is most highly expressed in adipose tissue and skeletal muscle, the major insulin-responsive tissues (Hu et al., Proc Natl Acad Sci USA, 1994, 91, 3730-3734; Tsukiyama-Kohara et al., Genomics, 1996, 38, 353-363). The human gene maps to chromosome 8p12 (Tsukiyama-Kohara et al., Genomics, 1996, 38, 353-363). The mouse eIF4E-BP1 gene consists of three exons, spans approximately 16 kb and maps to mouse chromosome 8 (Tsukiyama-Kohara et al., Genomics, 1996, 38, 353-363).
Rather than preventing the binding of eIF4E to mRNA caps, eIF4E-BP1 prohibits the binding of eIF4E to eIF4G, thereby preventing formation of a complex that is necessary for efficient binding and proper positioning of the 40S ribosomal subunit on the target mRNA. The region to which eIF4E binds is a common motif shared by eIF4G and eIF4E-BP1, and point mutations in this region of eIF4E-BP1 abolish binding to eIF4E (Mader et al., Mol Cell Biol, 1995, 15, 4990-4997). eIF4E-BP1 exists in a 1:1 ratio with eIF4E, and eIF4E-BP1 and eIF4G bind to eIF4E in a mutually exclusive manner (Rau et al., J Biol Chem, 1996, 271, 8983-8990).
Phosphorylation of bp 1 results in the release of eIF4E, allowing the formation of the eIF4F complex and eIF4F-dependent translation (Lin et al., Science, 1994, 266, 653-656; Pause et al., Nature, 1994, 371, 762-767). Two motifs are required for the efficient phosphorylation of eIF4E-BP1; the RAIP motif, which is found in the NH2-terminal region of EIF4E-BP1 and the TOS motif, which is formed by the last five amino acids of eIF4E-BP1 (Schalm and Blenis, Curr Biol, 2002, 12, 632-639; Tee and Proud, Mol Cell Biol, 2002, 22, 1674-1683). Mitogen-activated protein kinase, the major insulin-stimulated kinase in rat adipocytes, can phosphorylate recombinant eIF4E-BP1 in vitro (Lin et al., Science, 1994, 266, 653-656). However, MAP kinase did not readily phosphorylate eIF4E-BP1 when complexed with eIF4E. Moreover, the immunosuppressant rapamycin, which blocks activation of the kinase p70s6K by insulin without affecting the activation of MAP kinase, attenuated the stimulation of eIF4E-BP1 phosphorylation by insulin and significantly inhibited the dissociation of eIF4E-BP1 from eIF4E, without affecting MAP kinase activity. Furthermore, a MAP kinase kinase inhibitor markedly decreased insulin-stimulated MAP kinase activity without affecting eIF4E-BP1 phosphorylation or association with eIF4E (Lin et al., J Biol Chem, 1995, 270, 18531-18538). The inhibitory target of rapamycin, FRAP/mRAFT/mTOR, is an upstream element of the p70 signaling pathway, thus inhibition of eIF4E-BP1 phosphorylation by rapamycin is mediated by the mTOR signaling pathway, independently of MAP kinase (von Manteuffel et al., Proc Natl Acad Sci USA, 1996, 93, 4076-4080). The phosphorylation of eIF4E-BP1 by mTOR on threonine-36 and threonine-45 in vitro prevented the binding of eIF4E-BP1 to eIF4E (Brunn et al., Science, 1997, 277, 99-101; Burnett et al., Proc Natl Acad Sci USA, 1998, 95, 1432-1437). mTOR activity is required for the phosphorylation of eIF4E-BP1 in insulin-stimulated human embryonic kidney cells, and threonine-45 appears to be the major regulator of the in vivo interaction between eIF4E-BP1 and eIF4E (Brunn et al., Science, 1997, 277, 99-101; Burnett et al., Proc Natl Acad Sci USA, 1998, 95, 1432-1437).
Intracellular nutrients, as well as extracellular growth factors, also utilize eIF4E-BP1 as an effector of a signaling network. With respect to the availability of nutrients, the responsiveness of eIF4E-BP1 to insulin requires only the presence of amino acids, unlike other translational regulators which also require glucose (Campbell et al., Biochem J, 1999, 344 Pt 2, 433-441). The addition of leucine to isolated rat adipocytes significantly stimulated eIF4E-BP1 phosphorylation and leptin secretion in a rapamycin-sensitive and actinomycin D-resistant manner, indicating that leucine activates translation of leptin mRNA through the mTOR/bp pathway (Roh et al., Am J Physiol Endocrinol Metab, 2003, 284, E322-330). Leptin is produced mainly by adipose cells and regulates food intake and whole body energy balance, and because insulin levels respond to the nutritional status of the body, insulin has been suggested as a potential mediator between food intake and leptin production. The finding that leucine stimulates mTOR/eIF4E-BP1-mediated leptin production provides a possible connection between nutrient intake and circulating leptin levels (Roh et al., Am J Physiol Endocrinol Metab, 2003, 284, E322-330). An additional example of a link between nutritional status and translation is seen in skeletal muscle, a tissue where eIF4E-BP1 is abundantly expressed. In muscle from fasted rats, the amount of eIF4E associated with eIF4E-BP1 (and thus inhibited) is increased 5-fold as compared to muscle from freely fed animals. One hour following refeeding of a nutritionally complete diet, eIF4E-BP1 phosphorylation is increased, and the amount of eIF4E-BP1 bound to eIF4E is lowered to freely fed control values (Yoshizawa et al., Biochem Biophys Res Commun, 1997, 240, 825-831).
Systemic disruption of mouse eIF4E-BP1 does not lead to any abnormalities in the development or reproductive behavior of female mice, but does cause a 10% reduction in the body weight of male mice. The expression of eIF4E-BP1 and eIF4E in these mice does not appear to be altered (Blackshear et al., J Biol Chem, 1997, 272, 31510-31514). Surprisingly, a subsequent systemic gene disruption in a different mouse strain demonstrated that the interaction between bp1 and eIF4E impacts body weight, and fat and glucose metabolism. The bp1-deficient mice display reductions in fat tissue growth and weight gain, and also exhibit decrease circulating leptin levels. Furthermore, the eIF4E-BP1-deficient mice are hypoglycemic, suggesting that eIF4E-BP1 gene disruption can modulate insulin signaling. The mice bearing the eIF4E-BP1 disruption have a higher metabolic rate, which could be associated with the replacement of white fat tissue with brown fat tissue, which contains an uncoupling protein that generates heat by circumventing the mitochondrial proton battery. These results demonstrate that cap-dependent translation, in which eIF4E-BP1 functions as an important modulator, significantly regulates energy homeostasis and glucose and fat metabolism (Tsukiyama-Kohara et al., 2001, Nature Med. 7, 1128-1132; Sonenberg et al., 2003).
In some instances, the association of eIF4E-BP1 with eIF4E is stimulated. Agents that raise cyclicAMP levels increase the amount of eIF4E bound to eIF4E-BP1 and attenuate the effects of insulin on eIF4E-BP1 (Lin and Lawrence, J Biol Chem, 1996, 271, 30199-30204). Certain viruses, such as encephalomyocarditis virus and polio virus, promote the association of eIF4E-BP1 with eIF4E, thereby inhibiting translation of the capped mRNA of the host cell while allowing viral protein synthesis (Gingras et al., Proc Natl Acad Sci USA, 1996, 93, 5578-5583).
Induction of cellular differentiation and reduction of cellular proliferation are concomitant with a reduction in translation rates, as is observed during human myeloid cell differentiation. When induced to differentiate into monocytes/macrophages, cells from the HL-60 promyelocytic leukemia cell or U-937 monoblastic cell lines exhibit a decrease in the phosphorylation of eIF4E-BP1. In contrast, when HL-60 cells are stimulated to differentiate into granulocytic cells, the amount of eIF4E-BP1 is decreased, whereas phosphorylation of eIF4E-BP1 is not affected. Conversely, bp2 levels are markedly increased. These findings suggest that translation machinery is differentially regulated during human myeloid cell differentiation (Grolleau et al., Leukemia, 2000, 14, 1909-1914).
The disregulation of signaling networks that promote cell proliferation is often observed in association with cancer (Lawrence and Abraham, Trends Biochem Sci, 1997, 22, 345-349). Expression of excess eIF4E-BP1 in cells transformed by eIF4E or v-src results in significant reversion of the transformed phenotype, demonstrating that eIF4E-BP1 can function as an inhibitor of cell growth (Rousseau et al., Oncogene, 1996, 13, 2415-2420). US Patent Application Publication US 2003/0144190 A1 (Sonenberg et al) describes methods of immortalizing an oncogene-induced transformed cell which comprise increasing the amount of eIF4F pre-initiation complex by desequestration and/or inhibition of the sequestration of eIF4E in a complex with an eIF4E sequestering agent that comprises an antisense RNA complementary to the nucleotide sequence encoding for 4E-BP1.
Given the link between eIF4E-BP1-regulated translation initiation and food intake, and the importance of bp1 in regulating energy homeostasis, glucose metabolism and fat metabolism, it is of value to identify specific inhibitors of eIF4E-BP1. Currently, there are no known therapeutic agents that target eIF4E-BP1. Consequently, there remains a long felt need for agents capable of effectively inhibiting eIF4E-BP1. Antisense technology is an effective means of reducing the expression of specific gene products and therefore is uniquely useful in a number of therapeutic, diagnostic and research applications for the modulation of eIF4E-BP1 expression.
The U.S. Pat. No. 6,410,715 describes a purified human nucleic acid sequence encoding a cellular component that binds to eIF4E comprising a coding sequence for the protein eIF4E-BP1, and discloses a method for screening a non-hormone agent potentially useful to treat a hormone disorder (Sonenberg et al., 2000).
The US pre-grant publication 2003/0041341 (Sonenberg et al., 2003) discloses a method of decreasing fat tissue growth and/or weight gain, comprising administering an agent which desequesters eIF4E from a sequestering agent, wherein said sequestration of eIF4E is through its interaction with eIF4E-BP1, and wherein said desequestration or inhibition of sequestration is effected by an inhibition of the synthesis of eIF4E-BP1, comprising an agent which inhibits the synthesis of eIF4E-BP1, wherein said agent comprises an antisense RNA complementary to the nucleotide sequence encoding for eIF4E-BP1. Described therein are also methods of treating obesity and diabetes, comprising administering an agent which increases the amount of eIF4E available for a formation of eIF4F preinitiation complex, wherein said agent is an agent which desequesters eIF4E from eIF4E-BP1. Disclosed in this application is an antisense oligonucleotide primer targeting mouse eIF4E-BP1, and generally disclosed are oligonucleotide primers at least 12 nucleotides in length, preferably between 15 and 24 nucleotides.
The present invention provides compositions and methods for inhibiting eIF4E-BP1 expression.